Gastrointestinal cancer is a significant health problem, ranking fourth in incidence and second in death in the United States. Abnormal differentiation and increased proliferation are hallmarks of carcinogenesis. The molecular mechanisms that regulate cellular proliferation and differentiation in gastrointestinal development are far from being understood completely. We have shown that by gene targeting that the transcription factor Fkh6 (Forkhead homologue 6) is essential in the control of proliferation in the gastrointestinal epithelium. Fkh6, however, is expressed solely in the mesenchyme of the gastrointestinal tract. While the molecular mechanisms by which Fkh6 regulates gastrointestinal proliferation are unknown, we hypothesize that Fkh6 impacts on a signal transduction pathway from the mesenchyme to the epithelia of the gastrointestinal tract. Specific Aims of this proposal include the detailed characterization of the role of Fkh6 in gastrointestinal development. First, the site of action of Fkh6 in the gastrointestinal tract will be determined through the generation of chimeras between the wild type and Fkh6-/- embryos. Second, we will investigate whether Fkh6 affects cell migration in the intestinal epithelium, as this might contribute to the observed lengthening of the villi in the Fkh6-/- mice. Third, we will analyze the role of the Zinc-finger protein GKLF (Gut Kruppel Like Factor), in gastrointestinal development by loss of function and gain of function experiments. GKLF is expressed in the gastrointestinal epithelium and has been shown to inhibit proliferation in transfection experiments. GKLF is an gastrointestinal epithelium and has been shown to inhibit proliferation in transfection experiments. GKLF is an excellent candidate for one of the mediators of the Fkh6 controlled signal cascade, as GKLF mRNA is reduced in the Fkh6 mutants. Fourth, we will undertake an analysis of strain differences in the severity of the Fkh6-/- phenotype. This effort is based on the observation that the severity of the Fkh6 phenotype depends to a large extent on the genetic background. The screen will identify one or modifier loci that impact on the signaling pathway controlled by Fkh6. Together, these studies will further our understanding of the regulatory circuits that control gastrointestinal proliferation in normal development and carcinogenesis.